This invention relates to a process for preparing a calcium-dependent oxygenase.
More particularly it relates to a process for preparing an enzyme having a continuously photogenic performance when the enzyme is continuously added to a substrate, by treating the protein part of a Ca-binding photoprotein aequorin with a reducing agent and a metal ion. 2. Description of the Related Art
A Ca-binding photoprotein aequorin existent in nature emits light within several seconds when coelenterazine as an emitter present in the protein is oxidized in the presence of trace Ca ion. Since this series of reactions is irreversible, it is difficult to maintain the light-emission continuously and for a long time.
In order to solve the above-mentioned problem, the present inventor has performed extensive research employing the techniques of gene engineering and protein engineering.
If an apoaequorin(protein part) obtained from natural aequorin and an apoaequorin prepared according to a recombinant DNA technique (see Japanese patent application Nos. Sho 60-280259/1985, Sho 61-249098/1986, Sho 61245108/1986, Sho 61-245109/1986, Sho 62-126373/1987 and Sho 62-126374/1987) can be subjected to enzyme reaction for a long time, as in the case of conventional enzymes, then the general-purpose properties of the apoaequorin in the aspect of detection method are enhanced as compared with the above-mentioned instantaneously photogenic aequorin and it is possible to detect continuous light-emission by means of X-ray film, etc. This makes it possible to utilize the aequorin as a substitute for radioisotope; hence it has a utility.
The gist of the present invention consists in utilizing the catalytic function of the protein part (apoaequorin) of the Ca-binding photoprotein aequorin i.e. a function at which a substrate coelenterazine is decomposed and emits light by the addition of Ca.sup.2+ in the presence of oxygen.
As to natural aequorin or aequorin prepared through recombinant DNA, apoaequorin-oxygen-coelenterazine is present in a complex form and this instantaneously emits light by means of Ca.sup.2+ ion. This reaction is irreversible and when a substrate is added and also a reducing agent is added, only a very weak emitted light can be detected; hence it has a poor utility.
The present inventor further has performed extensive research on a treating method with a reducing agent taking into account the change in the higher order structure of apoaequorin, addition of Ca.sup.2+ ion, etc. and a process of preparing an apoaequorin having a sufficient utility i.e. a Ca-dependent oxygenase which catalyzes a continuous light emission.
As a result, we have found that the above-mentioned problems can be solved by a Ca-dependent oxygenase obtained by treating apoaequorin with a reducing agent in a suitable concentration to obtain a reduction type apoaequorin and reacting this apoaequorin with a metal ion preferably in the presence of a substrate coelenterazine.